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The endochitinase gene (chi3023) of Bacillus thuringiensis (Bt) serovar morrisoni strain 3023 was amplified via polymerase chain reaction (PCR) and cloned in Escherichia coli. | Turk J Biol 35 (2011) 1-7 © TÜBİTAK doi:10.3906/biy-0905-5 Molecular cloning, characterization, and homologous expression of an endochitinase gene from Bacillus thuringiensis serovar morrisoni Sezer OKAY, Gülay ÖZCENGİZ Department of Biological Sciences, Middle East Technical University, 06531, Ankara - TURKEY Received: 20.05.2009 Abstract: The endochitinase gene (chi3023) of Bacillus thuringiensis (Bt) serovar morrisoni strain 3023 was amplified via polymerase chain reaction (PCR) and cloned in Escherichia coli. The ORF of chi3023 (GenBank Accession Number: DQ993175) consists of 2031 nucleotides encoding a 676-residue protein with a calculated molecular mass of 74.5 kDa and a pI value of 6.0. The amino acid sequence of Chi3023 was compared with previously sequenced Bt chitinases and the phylogenetic relationships among them were determined. The deduced N-terminal 34 amino acids of the premature Chi3023 exhibited a typical signal peptide. The E. coli-Bt shuttle vector pHT315 was used for homologous expression of chi3023. Introduction of recombinant pHT315BTC, carrying chi3023 into Bt serovar morrisoni 3023, resulted in a 23-fold increase in endochitinase activity (0.185 U/mg versus 4.256 U/mg). Key words: Bacillus thuringiensis, characterization analysis, endochitinase, gene cloning Bacillus thuringiensis serovar morrisoni endokitinaz geninin moleküler kopyalanması, karakterize edilmesi ve homolog ifadesi Özet: Bacillus thuringiensis (Bt) serovar morrisoni 3023 suşuna ait endokitinaz geni (chi3023) polimeraz zincir reaksiyonu (PCR) ile çoğaltılıp E. coli’ye kopyalandı. chi3023 geni (Gen Bankası Erişim Numarası: DQ993175), 676 aminoasit kodlayan 2031 nükleotitten oluşmaktadır. Moleküler ağırlığı 74.5 kDa, pI değeri de 6.0 olarak hesaplandı. Chi3023’ün aminoasit dizisi daha önce dizilenmiş Bt kitinazları ile karşılaştırıldı ve aralarındaki filogenetik ilişki belirlendi. Prematüre Chi3023’ün N-ucundaki 34 aminoasit, tipik sinyal peptidi özelliği göstermektedir. .
